1. Field of the Invention
The present invention relates generally to the fields of molecular and cellular biology. More particularly, it concerns compositions and methods for removal of nucleic acid probes from sample nucleic acids, for example, sample nucleic acids attached to a solid support. The invention also concerns methods of stripping and reusing nucleic acid blots.
2. Description of Related Art
The central dogma of molecular biology holds that genetic information flows from nucleic acids (DNA and RNA) to proteins, and that the functions and relative abundances of proteins within a biological system defines that system. Proteins are polymers of amino acids and it is the sequence of amino acids within the polymer that determines the function of the protein. Amino acid sequences are encoded by nucleic acid sequences that exist in domains known as genes that are present in the genomes of biological entities. Genomes are the nucleic acid storehouses of genetic information present in all known living systems.
Genomes are typically composed of DNA, though there are a number of viruses with RNA genomes. The genes in DNA are converted to proteins via an RNA intermediate known as messenger RNA (mRNA). The rate of conversion of a DNA segment composing a gene to mRNA, and the subsequent degradation rate of that mRNA, affects protein production which ultimately impacts the state of that organism. The importance of nucleic acids in biology is unquestioned, thus a substantial amount of research effort has been applied to identifying gene sequence, structure, and function, as well as the relative rates of messenger RNA synthesis and degradation and the abundance of particular mRNA species.
A variety of techniques have been employed in the detection and quantification of both RNA and DNA. Most of these techniques rely on the capacity of nucleic acids to interact in a sequence specific manner known as hybridization. Hybridization occurs when two nucleic acid molecules possessing complementary sequences interact to form a, typically, double helical structure. Hybridization studies designed to identify or quantify a sample nucleic acid possessing a given sequence typically involve synthesizing an isotopically or non-isotopically labeled nucleic acid probe with a sequence that is complementary to the sample nucleic acid to be detected. The labeled nucleic acid probe is incubated with the sample population to allow hybridization between target and probe. Labeled nucleic acids that have not hybridized are removed, leaving the hybridized probe to be detected via the isotopic or non-isotopic label.
Of the many techniques for nucleic acid analysis that rely on hybridization, some involve immobilizing the sample nucleic acid on a solid support. Detection and/or quantification methods that rely on immobilization of RNA or DNA species by physical attachment to a solid support are well-known and include, but are not limited to, Northern (RNA sample nucleic acid), Southern (DNA sample nucleic acid), dot, slot, colony lifts, and related blot analyses (Maniatis et al., 1989), sandwich hybridization (Kwoh et al., 1989), and hybridization of a labeled amplification product to an oligonucleotide attached to a solid support (Woolford and Dale 1992).
In blot analyses, the solid support is usually a membrane and the sample is usually a heterogeneous population of nucleic acids purified from a cell culture, tissue, or organism. UV or chemically induced crosslinking between the sample and membrane generates the sample matrix. Isotopically or non-isotopically labeled probes (nucleic acids possessing sequences complementary to the RNA or DNA to be detected) are synthesized by enzymatic or synthetic means and mixed with the sample matrix. Hybridization of the probe to the subset of nucleic acids with complementary sequences, removal of the non-hybridized probe molecules by extensive washing, and detection of the remaining label provides for the positive identification and quantification of nucleic acids possessing the given sequence.
The sample matrices can be used multiple times, provided that all hybridized probe is removed prior to initiating hybridization of a new probe. The complete removal of the preceding probe is important, as the signal from one probe can affect the detection and quantification of a second nucleic acid. Removing any hybridized nucleic acid requires that sufficient energy be introduced to disrupt all hydrogen bonding and stacking interactions between probe and target. Additionally, the solution must be stringent enough to deny reannealing once the interaction between probe and target has been disrupted. Temperatures that exceed the melting temperature of the probe and target in solutions that lack monovalent salts and include detergents are typically used to remove probes from a solid support (Maniatis et al., 1989). However, in many instances, this level of stringency is inadequate to completely remove the probe leaving residual signal that can affect the subsequent analysis of other targets. In addition, the extreme conditions often cause irreversible damage to the sample matrix, either by removing sample nucleic acids altogether, or altering their chemical properties. These two problems often necessitate that a sample matrix be used one time and then discarded.
Given the time and expense involved in the preparation of a sample matrix, in addition to the fact that some sample matrices would be difficult to reproduce given the unavailability of starting materials (for example, patient samples, etc.), the ability to reuse a sample matrix would represent a significant advance in the art.
The present invention overcomes the limitations present in the art by providing compositions and methods for removing hybridized nucleic acid probes from sample nucleic acids, for example from sample matrices consisting of sample nucleic acids attached to solid supports. The invention provides for the specific degradation or cleavage of a nucleic acid probe following hybridization and detection, reducing the energy of hybridization between the sample nucleic acid and the various nucleic acid probe molecules. The reduction in hybridization energy provides for complete removal of the nucleic acid probe under conditions that are less likely to affect the sample nucleic acids and/or sample matrix (lower temperature and/or less extreme conditions). The present invention thus provides for the reuse, and even multiple uses, of sample matrices. The present invention also provides kits, including, but not limited to, kits for nucleic acid probe degradation, cleavable nucleic acid probe synthesis, sample matrix stripping and reuse, and nucleic acid detection.
For clarity, the nucleic acid(s) attached to a solid support will be referred to as the sample nucleic acid(s) or target; the solid support plus sample nucleic acid(s) will be referred to as the sample matrix; and the nucleic acid probe (or xe2x80x9cprobexe2x80x9d) will be the nucleic acid molecule that hybridizes to the sample nucleic acid(s).
Various basic aspects of the invention are summarized as follows. Note that, in accordance with long-standing patent law practice and convention, the words xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d denote xe2x80x9cone or morexe2x80x9d when used in this application, including the claims. Further, the breaking of a first bond in a nucleic acid probe can occur in conjunction with the breaking of other bonds, and there is no limitation in the invention as to breaking of xe2x80x9cone and only one bond.xe2x80x9d
The invention provides a method of removing a nucleic acid probe from a sample nucleic acid comprising obtaining a sample nucleic acid and a nucleic acid probe, the nucleic acid probe associated with the sample nucleic acid, breaking at least a first bond of the nucleic acid probe, and removing the nucleic acid probe from the sample nucleic acid. Of course, more bonds than a first bond may be broken, and, typically, more than one bond will be broken.
In certain embodiments, the sample nucleic acid probe comprises DNA. In other embodiments, the sample nucleic acid probe comprises RNA. In still other aspects, the nucleic acid probe comprises DNA and RNA.
In embodiments of the present invention, the first bond on the nucleic acid probe is broken via chemical or physical means. The nucleic acid probes of the present invention may be comprised of a variety of different types of degradable phosphate backbone bonds that are broken. In certain aspects of the invention, the bond is a phosphodiester bond. In alternative embodiments, the bond is a phosphorothioate bond. The bond may be broken by an enzyme, exemplified by, but not limited to, uracil DNA glycosylase, a ribonuclease, inosine ribonuclease, deoxyribonuclease, or combinations thereof. As used herein in various embodiments of the present invention, the term xe2x80x9cnucleasexe2x80x9d will be understood to include ribonuclease, deoxyribonuclease, endonuclease and exonuclease. In other embodiments, the bond is broken by one or more particular wavelengths of light, or by temperature. In further preferred embodiments, the degradable bond of the nucleic acid probe is broken by a combination of two or more of the above methods.
In particular aspects of the invention, the sample nucleic acid comprises DNA. In other aspects, the sample nucleic acid comprises RNA. In certain embodiments, the sample nucleic acid is comprised within a cell. In further embodiments, the sample nucleic acid is comprised within a virus.
In various preferred aspects of the present invention, the sample nucleic acid is attached to a solid support. In certain embodiments, the solid support is a membrane, exemplified by, but not limited to, a nitrocellulose membrane or a nylon membrane. In other embodiments, the solid support is a resin, including, but not limited to, an ion exchange chromatography resin such as an anion or cation exchange resin, or an affinity chromatography resin. In further embodiments, the solid support is plastic, such as a microtiter plate. Other examples of solid supports contemplated for use in the present invention include, but are not limited to, a magnetic bead, glass, or a microchip.
In particular embodiments, the sample nucleic acid is separated by electrophoresis prior to attachment to the solid support. In some embodiments, the sample nucleic acid may be cleaved by an enzyme prior to separation by electrophoresis.
In certain preferred aspects of the invention, the obtaining may be characterized as comprising obtaining a sample nucleic acid, obtaining a nucleic acid probe, and admixing the nucleic acid probe with the sample nucleic acid, for a period of time and under conditions sufficient to allow association of the nucleic acid probe with the sample nucleic acid.
The invention also provides a method of stripping a nucleic acid probe from a sample nucleic acid, the sample nucleic acid attached to a solid support, that may be characterized as comprising obtaining a solid support comprising a sample nucleic acid attached thereto, obtaining a nucleic acid probe, the nucleic acid probe comprising at least a first phosphorothioate bond, admixing the nucleic acid probe with the solid support, for a period of time and under conditions sufficient to allow association of the nucleic acid probe with the sample nucleic acid attached to the solid support, cleaving the phosphorothioate bond of the nucleic acid probe with iodine, removing the nucleic acid probe from the sample nucleic acid, and admixing sodium thiosulfate with the solid support, thereby removing excess iodine from the solid support.
The invention also provides a kit for removing a nucleic acid probe from a sample nucleic acid, comprising in a suitable container a compound that breaks at least a first bond of the nucleic acid probe. In certain aspects, the compound is a chemical, such as iodine. In other aspects, the compound is an enzyme, such as uracil DNA glycosylase, a ribonuclease or a deoxyribonuclease.
In certain embodiments of the kits of the present invention, the kit further comprises at least a first cleavable nucleotide for incorporation into the nucleic acid probe. In particular aspects, the cleavable nucleotide is a phosphorothioate nucleotide. In other aspects, the cleavable nucleotide is a uracil nucleotide. In further embodiments, the cleavable nucleotide is an inosine nucleotide.
Additionally, the present invention provides a kit for removing a nucleic acid probe from a sample nucleic acid, comprising, in a suitable container, probe degradation buffer, and reconstitution buffer. In certain preferred embodiments, the probe degradation buffer comprises iodine.
In other embodiments, the kits of the present invention further comprises, in a suitable container, at least a first cleavable ribonucleoside triphosphate, ATP, CTP, GTP and UTP, RNA polymerase, RNase inhibitor, and transcription buffer. In still other aspects, the kits further comprise, in a suitable container, at least a first cleavable deoxyribonucleoside triphosphate, dATP, dCTP, dGTP, dTTP and DNA polymerase. As used herein in certain aspects of the present invention, the term xe2x80x9cnucleotide(s)xe2x80x9d will be understood to include ribonucleotide(s) and deoxyribonucleotide(s).
Thus, in a particular embodiment, the present invention also provides a kit for removing a nucleic acid probe from a sample nucleic acid, that may be characterized as comprising, in a suitable container, probe degradation buffer comprising iodine and SDS, reconstitution buffer comprising sodium thiosulfate and SDS, [xcex1-S] CTP, ATP, GTP and UTP, an RNA polymerase selected from the group consisting of SP6 RNA polymerase, T7 RNA polymerase and T3 RNA polymerase, placental RNase inhibitor, and transcription buffer comprising sodium chloride, Tris (pH 8), magnesium chloride, spermidine-HCl, and DTT.
The present invention further provides a kit for detecting the association of a nucleic acid probe with a sample nucleic acid, comprising in a suitable container a solid support and a compound that breaks at least a first bond of the nucleic acid probe.
Components of the above-summarized particular aspects of the invention will be discussed in detail below. Of course, this invention is in no way limited to the particular embodiments of the invention summarized herein. Rather, the disclosure of this specification is more than adequate to enable the full scope of the invention to those of skill.
I. Nucleic Acid Probes
The nucleic acid probes used in most detection methods hybridize to their targets, typically, over regions in excess of one hundred nucleotides, although this length is not always required. The hybridization energy of long stretches is significant and requires substantial energy to disrupt. Breaks introduced in the polynucleotide chain of the probe molecule effectively reduce the energy required to disrupt interactions between probe and target by reducing the size of the regions of interaction. For instance, a probe that is 500 nucleotides long can be reduced to a series of polymers 5-10 nucleotides in length by periodically cleaving phosphodiester bonds along the polynucleotide backbone. xe2x80x9cMelting temperaturexe2x80x9d is a temperature that will cause hybridization of nucleic acids of a particular length to disassociate.
The melting temperature for an average ten nucleotide long probe is less than 50xc2x0 C., whereas that of a 500 nucleotide long probe exceeds 100xc2x0 C. Owing to this change in melting temperature, removing the short polymers from a target requires a relatively non-stringent wash of 50-60xc2x0 C., whereas boiling the sample matrix is often insufficient to remove a 500 nucleotide long probe. The specific degradation of the probe reduces the stringency required to remove the probe from a sample matrix and enhances its complete removal, thus facilitating the multiple usage of the sample matrix.
A. Size and Composition
Nucleic acid probes used for detecting targets attached to a solid support are typically 100 to 5,000 nucleotides long, although probes as short as ten nucleotides to probes as large as 1,000,000 nucleotides can be used provided appropriate conditions. The present invention thus also contemplates the use of nucleic acid probes of any intermediate length, including, but not limited to, probes of 20, 30, 50, 75, 150, 200, 300, 500, 600, 800, 1000, 2500, 7000, 10,000, 50,000, 100,000, 250,000, 500,000 or 750,000 nucleotides in length. The present invention involves cleaving the probe at positions held by a particular nucleotide (for instance, cytidine), so that, regardless of size, the probe is reduced to constituent fragments that can be easily removed from the sample matrix. Nucleic acid probes are typically composed of RNA or DNA, although a probe comprising both RNA and DNA could also be used. The invention is applicable for probes of each these compositions.
Another nucleic acid molecule that is contemplated for use as a probe is a peptide nucleic acid (PNA). PNAs can be used as degradable probes. PNA probe removal can be facilitated by digestion with a protease that would degrade the peptide bonds of the backbone without affecting the nucleic acids attached to the solid support. The resulting peptide nucleic acid fragments can be easily removed by a mild wash.
B. Degradable Probes Several methods exist for producing nucleic acids that are more labile under a given set of conditions. These can be employed in the invention. Applications for specifically degradable nucleic acids have been described in the literature. In the few cases where the described use for the technology is probing a nucleic acid on a solid support, the purpose for cleaving the probe is to release the reporter into the solution to enhance the sensitivity of the assay and to avoid background caused by non-specific interactions of probe with the solid support (Urdea, 1995; Urdea and Horn, 1995; Urdea and Horn, 1996a; Urdea and Horn, 1996b). Other applications for making specifically degradable nucleic acids are cloning (Mag et al., 1991; Rashtchian and Berninger, 1992); producing amplification products that can be specifically degraded following detection to decrease the risk of contamination (Longo et al., 1990; Frasier et al., 1996); analyzing therapeutic oligonucleotides (Wyrzykiewicz, U.S. Pat. No. 5,629,150); sequencing (Gish and Eckstein, 1988); and footprinting interactions of molecules with nucleic acids (Schatz et al., 1991). These degradable probe technologies apparently have not been used to facilitate the stripping of blots.
Effective degradable nucleic acid probes typically share several characteristics. The element of the probe that confers cleavage should not significantly affect the hybridization kinetics, energy, or specificity between the probe and target. The degradation reaction advantageously occurs regardless of whether the probe is hybridized to a complementary sequence. The level of degradation should be sufficient to reduce nucleic acid polymers of hundreds or thousands of nucleotides to polymers of smaller size, typically less than one hundred nucleotides. The reagents and wash conditions required to degrade and remove the probe from the sample matrix should have minimal impact on the sample nucleic acid or its associate with a solid support.
Methods for the specific degradation of nucleic acid probes are exemplified by but not limited to: enzymatic degradation resulting from specific recognition of the probe but not the sample nucleic acid; chemical degradation resulting from the presence of chemical groups in the probe that react with agents added to a sample matrix that result in strand scission of the probe but not sample nucleic acid; light or temperature induced degradation resulting from the presence of chemical groups in the probe that are activated by light or extreme temperatures to cause strand scission of the probe but not the sample nucleic acid; and combinations of any two or more methods, for example the combination of enzymatic and chemical degradation.
In some embodiments, the internucleotide phosphates of RNA or DNA can be modified by replacing one of the non-bridging oxygens with a phosphothiol or phosphodiester bond. Such modification has little effect on the hybridization characteristics of the molecule but has a significant impact on its chemistry. Thiol-modified phosphates (phosphorothioates) react with iodine and related reagents to cause strand scission of the modified nucleic acids (Gish and Eckstein, 1988). Thus the existence of phosphorothioates within a nucleic acid (RNA or DNA) can provide a method for the specific reduction of that nucleic acid to smaller polymers.
In embodiments where the nucleic acid probe comprises at least a first phosphorothioate bond, the bond may be broken by a chemical, such as iodine. In certain aspects, the concentration of the iodine is between about 5 xcexcM and about 500 mM. In preferred embodiments, the concentration of the iodine is between about 0.1 mM and about 25 mM. In more preferred embodiments, the concentration of the iodine is between about 0.5 mM and about 2 mM. While the concentrations of iodine employed to break a phosphorothioate bond are described in discrete ranges, it will be understood by the person of ordinary skill in the art that intermediate ranges of iodine concentration are also encompassed within these ranges. Thus, the iodine concentration may be between about 5 xcexcM and about 250 mM, between about 25 xcexcM and about 500 mM, between about 50 xcexcM and about 100 mM, about 0.1 mM and about 10 mM, between about 0.2 mM and about 25 mM, between about 0.25 mM and about 5 mM, between about 0.5 mM and about 1 mM, between about 1 mM and about 2 mM, or between about 0.75 mM and about 1.5 mM.
Additional methods exist for producing nucleic acids that are particularly susceptible to degradation under specific conditions. These can be incorporated into the invention. For example, the enzyme uracil DNA glycosylase removes the uracil base from the sugar-phosphate backbone, leaving an abasic site. This abasic site is recognized and cleaved by certain nucleases, for example, Exonuclease IV (Rashtchian and Berninger, 1992) and Exonuclease I (Brody, 1991) from E. coli. Thus, the combination of uracil DNA glycosylase and an appropriate nuclease serves to cleave the phosphodiester backbone of nucleic acid probes which have uridine incorporated therein. The abasic sites created by the action of uracil DNA glycosylase can also be cleaved by acid or base hydrolysis. Thus the combination of enzymatic and chemical methods are also contemplated for use in cleaving nucleic acid probes.
In one aspect of the present invention, the nucleic acid probe comprises at least a first uracil residue. Nucleic acid probes comprising one or more uracil residues may be broken by a combination of uracil DNA glycosylase and an exonuclease, or in alternate aspects, by a combination of uracil DNA glycosylase and a chemical that promotes either acid hydrolysis by lowering the pH, for example to a pH value below a neutral pH, including, but not limited to, a pH of 6, 5, 4, 3, 2, 1 or 0, and intermediate values thereof, or base hydrolysis by raising the pH, for example to a pH value above a neutral pH, including, but not limited to, 8, 9, 10, 11, 12, 13 or 14, and intermediate values thereof. In particular embodiments of the present invention, the bond is broken by a hydroxyl ion. In certain aspects, the concentration of the hydroxyl ion is between about 1 M and about 10xe2x88x926 M. In preferred embodiments, the concentration of the hydroxyl ion is between about 10xe2x88x921 M and about 10xe2x88x925 M. In more preferred embodiments, the concentration of the hydroxyl ion is between about 10xe2x88x923 M and about 10xe2x88x925 M. It will be understood by the person of ordinary skill in the art that intermediate ranges or values of hydroxyl ion concentration are also encompassed within these ranges.
Also, inosine RNase can be used to cleave RNA probes at positions occupied by inosine (Scadden and Smith, 1997). Additionally, RNA probes are labile in solutions possessing elevated hydroxyl levels (pH of about 9 or above) or ribonucleases, including, but not limited to, ribonuclease A, ribonuclease B, ribonuclease C, ribonuclease S, ribonuclease T1, ribonuclease T2, ribonuclease U1 and ribonuclease U2 (all of the above ribonucleases are available commercially, for example from Sigma Chemical Company, St. Louis, Mo.). Similarly, DNA probes can be degraded by deoxyribonucleases, including, but not limited to, deoxyribonuclease I and deoxyribonuclease II (all of the above ribonucleases are available commercially, for example from Sigma Chemical Company, St. Louis, Mo.). All of the above can be used within the scope of the invention.
Photolabile groups that can be incorporated to provide nucleic acid probes that are degraded by specific light emissions are provided in U.S. Pat. No. 5,430,136 (incorporated herein by reference in its entirety). Such photolabile groups also can be incorporated into the invention.
C. Preparation of Degradable Probes
Methods for the specific degradation of nucleic acid probes are exemplified by, but not limited to: enzymatic RNA synthesis by transcription, primer extension, replication, or non-templated polymerization that incorporates nucleotides that are labile under defined conditions for the purpose of specific degradation of the probe molecule, for example using SP6 RNA polymerase (Green et al., 1983) or T7 RNA polymerase (Tabor and Richardson, 1985); enzymatic DNA synthesis by primer extension, promoter-driven polymerization, or non-templated polymerization that incorporates nucleotides that are labile under defined conditions for the purpose of specific degradation of the probe molecule, for example by nick-translation (Kelly et al., 1970) or random priming (Feinberg and Vogelstein, 1983); chemical synthesis of RNA, DNA, or similar molecules that possess chemical groups that are labile under defined conditions for the purpose of specific degradation of the probe molecule (reviewed in Caruthers et al., 1987); and post synthesis modification of a nucleic acid probe by chemical, photochemical, or enzymatic means to introduce changes that are labile under defined conditions for the purpose of specific degradation of the probe molecule.
The preparation of specifically degradable nucleic acid can be accomplished by a variety of methods. Nucleotides and their analogs can be incorporated by RNA and DNA polymerases to provide nucleic acids with labile sites. RNA and DNA possessing labile nucleotides can also be chemically synthesized using phosphoramidites. Labile sites can even be introduced to RNA or DNA molecules following enzymatic or chemical synthesis. For example, adenosine deaminase converts adenosines to inosines within a nucleic acid molecule (Hough and Bass, 1994). This conversion effectively converts an otherwise stable RNA into a substrate for inosine ribonuclease providing an avenue for the specific destruction of the nucleic acid (Scadden and Smith, 1997).
D. Detectable Labels
Labeled nucleic acids are used to visualize the hybridized product that reveals the presence and/or amount of a target molecule. The label can be a radioisotope, an enzyme, or a fluorescent, chemiluminescent, or bioluminescent molecule. The labels can be either a component of the nucleotide (especially radioisotopes), covalently attached to a nucleotide, or attached to the nucleotide via a binding agent (for instance, an enzyme-linked antibody bound to a nucleic acid). The labeled nucleotide(s) can either be part of the region of the nucleic acid probe that hybridizes to the sample nucleic acid, or be part of a nucleic acid probe region that is not complementary to the sample nucleic acid. The label is preferably a part of the nucleic acid probe, but in certain embodiments, such as sandwich hybridization, the label can be incorporated into a distinct nucleic acid molecule that binds to a nucleic acid probe, particularly when the nucleic acid probe is hybridized to a sample nucleic acid attached to a sample matrix.
The detectable labels contemplated for use in the present invention are exemplified by, but not limited to: radioactive labels, such as tritium, carbon-14, phosphorus-32 or phosphorus-33 or sulfur-35; enzymatic labels, such as alkaline phosphatase or horseradish peroxidase; fluorescent labels, such as green fluorescent protein (GFP), rhodamine, fluorescein isothiocyanate, phycoerythrin, phycocyanin, allophyocyanin, o-phthaldehyde, fluorescamine, Texas Red or renographin; chemiluminescent labels, such as acridinium salt, isoluminol, imidazole, theromatic acridinium ester, luminol, or oxalate ester; bioluminescent labels, such as aequorin, luciferin, or luciferase; or metal labels, such as gold.
II. Sample Nucleic Acids
A. Size and Composition
Typical size ranges for nucleic acids attached to a solid support to generate the sample matrix are 8 to 20,000 nucleotides, though entire chromosomes (comprising approximately 109 base pairs) have been assayed using immobilization methods. The present invention thus also contemplates the use of sample nucleic acids of any intermediate length, including, but not limited to, 9, 10, 12, 15, 20, 30, 50, 75, 150, 200, 300, 500, 600, 800, 1000, 2500, 7000, 10,000, 50,000, 100,000, 250,000, 500,000, 750,000, 106, 5xc3x97106, 107 or 108 nucleotides in length. RNA and DNA in both single and double-stranded form can be used as the sample. Though purification is often a prerequisite for attachment to a solid support (for example Northern and Southern blotting, dot/slot blots, etc.), many methods actually utilize unpurified nucleic acids (for example colony or plaque lifts, in situ hybridization, etc.).
B. Sources
Virtually any source of nucleic acid can form a sample for immobilization in the creation of a sample matrix. Single-stranded and double-stranded RNA and DNA purified (either together or separately) from prokaryotic, eukaryotic, or viral sources can all be used. In addition, nucleic acids from in vitro enzymatic reactions or chemical syntheses can likewise be affixed to a solid support to form the sample matrix. Those of skill will be able to determine appropriate sources of such acceleration.
C. Isolation Methods
There are numerous methods known to those of skill for isolating nucleic acids from biological sources (reviewed in Maniatis, 1989). DNA and RNA isolation is routinely accomplished by homogenizing a sample to release all cellular or capsid components into solution. The nucleic acids are then purified away from the contaminating lipids, proteins, and small molecules by preferential binding to a solid support, organic extraction, or differential precipitation.
III. Solid Supports
Many different examples of solid supports for use in attachment of sample nucleic acids are known to those of skill in the art. The methods used in the attachment of sample nucleic acids to solid supports is also well known and understood by those of skill in the art (Maniatis et al., 1989; Ausubel et al., 1989).
A. Membranes
Nitrocellulose, nylon and cellulose are the most common membranes used for immobilizing DNA and RNA in analyzing samples by Northerns, Southerns, dot/slot blots, and colony/plaque lifts. Nitrocellulose is thought to immobilize nucleic acids through hydrophobic and electrostatic interactions. Baking a blotted sample can be used to increase the strength of the interaction between the nitrocellulose and the nucleic acid sample. Nylon membranes are charged providing extensive electrostatic interactions with the phosphate backbone of both RNA and DNA. UV irradiation and baking or drying can be used to actually cross-link activated groups on the membrane to the nucleic acids in the sample. Those of skill know a variety of other methods and technologies for affixing nucleic acids to membranes, including, but not limited to, chemical interactions, protein/chemical interactions, protein/protein interactions and sugar trans-esterification.
B. Other Solid Supports
Resins, plastics, magnetic beads, glass, and microchips can also be used as the solid support in certain embodiments of the present invention. The supports may be derivatized to include chemical moieties that will interact with RNA or DNA to provide immobilization through electrostatic or hydrophobic interactions, covalent attachment (Fodor et aL, 1991), or by protein or small molecule interactions between a molecule attached to the nucleic acid sample (such as streptavidin) and a molecule attached to the solid support (such as biotin) (Huang et al., 1996).
IV. Hybridization Methodology
Methods for using the degradable nucleic acid probes of the present invention are exemplified by, but not limited to: Northern blots; Southern blots; dot/slot blots; colony/plaque lifts; ELISAs; using sample nucleic acids as templates, to synthesize probes that can be hybridized to and easily removed from nucleic acids attached to a solid support; and sandwich hybridization schemes where the degradable nucleic acid hybridizes to a polynucleotide attached to a solid support and a second polynucleotide that possesses a detectable label. Of course, many modifications of these procedures are known to those of skill and contemplated by the invention.
A. Southern Blot
DNA may be size fractionated by electrophoresis through a gel, typically agarose, then transferred and immobilized on a membrane in such a manner that the relative positions of the fractionated DNA fragments are maintained (Southern, 1975). The resulting blot can then be mixed with a nucleic acid probe to identify the subset of the DNA population that possesses a sequence that is complementary to the probe.
B. Northern Blot
RNA (especially messenger RNA) may be size fractionated by electrophoresis through a gel, typically agarose then transferred and immobilized on a membrane in such a manner that the relative positions of the fractionated RNA are maintained (Alwine et al., 1977). The resulting blot can then be mixed with a nucleic acid probe to identify the subset of the RNA population that possesses a sequence that is complementary to the probe.
C. Dot/Slot Blot
An RNA or DNA sample is spotted onto a membrane and immobilized. The spotting of the sample can either be done by simply dropping an aliquot onto the membrane (Kafatos et al., 1979), or using a filtration device to spot the nucleic acids in a fixed pattern (Brown et al., 1983). The dot or slot blot is incubated with a nucleic acid probe to allow hybridization between the probe and target molecules on the blot possessing sequences complementary to the probe. Removal of non-hybridized probe and detection of the remaining label provide identification and quantification of the target molecules.
D. Colony/Plaque Lifts
Bacterial and other colonies as well as viral plaques can be grown on solid media, producing clonal populations of an organism. In a typical colony or plaque lift, the nucleic acids from the clones are transferred to a membrane by lysing the colony/plaque and placing a membrane on top of the medium (Grunstein and Hogness, 1975). The RNA/DNA adsorbs to the membrane and is subsequently immobilized generating a colony or plaque blot. A probe is introduced to the blot and hybridization reveals those colonies or plaques possessing RNA or DNA that is complementary to the added probe.
E. Sandwich Hybridization
Sandwich hybridization schemes involve attaching a sample nucleic acid, typically a synthetic oligonucleotide, to a solid support. The sequence of the attached nucleic acid is designed to be complementary to the sequence of a nucleic acid probe molecule to be detected and/or quantified out of a population of nucleic acids. A sample population is then incubated with the oligonucleotide/solid support sample matrix. Non-hybridized members of the sample population are removed. A labeled nucleic acid whose sequence is complementary to another portion of the nucleic acid probe molecule is added and allowed to hybridize. Non-hybridized, labeled nucleic acid is removed leaving only the labeled nucleic acid that is hybridized to those nucleic acid probe molecules that are themselves hybridized to the sample matrix. The label is then detected, providing an estimate of the amount of a nucleic acid probe molecule in nucleic acid population.
F. In situ Hybridization
In situ hybridization provides a method for the morphological localization of RNA or DNA molecules of a given sequence (reviewed in Hofler et al., 1988). Sections or whole mounts of an organism are fixed to preserve tissue and cellular morphology (this includes immobilizing nucleic acids within the sample). A probe is mixed with the preserved tissue. Hybridization and detection identifies locations within the organism, tissue, or cell that house RNA or DNA molecules possessing sequences complementary to the probe.
G. Reverse Blots
Reverse blots differ from those described above in that nucleic acids of a single sequence are attached to a solid support to yield the sample matrix and the heterogeneous population serves as the probe. The heterogeneous population may be labeled, either directly by chemical means or enzymatically by second strand synthesis incorporating modified nucleotides. The labeled population is incubated with the sample matrix and the non-hybridized, labeled molecules are removed by washing. The hybridized molecules are then detected, providing for the positive identification of those nucleic acids within the population that possess a given sequence.
A related technique is used to detect the nucleic acid products of an amplification reaction such as PCR(trademark). In this detection scheme, the sample matrix consists of a solid support (beads, microtiter plates, and microchips are a few of the supports that have been used) covalently attached to oligonucleotides (or polynucleotides) possessing a sequence that is complementary to the target amplification product. One skilled in the art will appreciate that there are a variety of detection methods. In a commonly used strategy, a labeled nucleotide is incorporated during the amplification reaction, providing labeled nucleic acids that will serve as probes. The double stranded nucleic acid amplification products are dissociated, mixed with the sample matrix, and incubated. Non-hybridized molecules are washed from the solid support, leaving the labeled, hybridized amplification products for detection.
V. Kits
All of the essential materials and reagents required for the various aspects of the present invention may be assembled together in a kit. A variety of kits are contemplated, including, but not limited to, nucleic acid probe degradation kits, degradable nucleic acid probe synthesis kits, blot stripping and reuse kits, nucleic acid detection kits as well as combinations of any or all of the above into larger combination kits.
The container means will generally include at least one vial, test tube, flask, bottle or other container means, into which, for example, the nucleic acid probe removal or synthesis formulations are placed, preferably, suitably allocated or aliquoted.
When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred. The components of the kit may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kit may have a single container means, or it may have distinct container means for each compound. In certain aspects, one or more solid supports used for attaching either sample or probe nucleic acids are also provided.
The kits of the present invention also will typically include a means for containing the components in close confinement for commercial sale such as, e.g., injection or blow-molded plastic or cardboard containers into which the desired components are retained. Additionally, instructions for use of the kit components is typically included.
Components for an RNA probe synthesis and degradation kit may include, but are not limited to, an RNA polymerase, such as SP6 RNA polymerase, T7 RNA polymerase, or T3 RNA polymerase, and a ribonuclease inhibitor, such as placental ribonuclease inhibitor, antibodies to ribonucleases that inhibit their activity or small molecule ribonuclease inhibitors, such as uridine-vanadate, 2xe2x80x2 CMP, 2xe2x80x2 UMP or oxyvanadium IV.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.